Download ANTIBACTERIAL AND SYNERGISTIC ACTIVITY OF ETHANOLIC AJWAIN (TRACHYSPERMUM

Innovare
Academic Sciences
International Journal of Pharmacy and Pharmaceutical Sciences
ISSN- 0975-1491
Vol 6, Issue 6, 2014
Original Article
ANTIBACTERIAL AND SYNERGISTIC ACTIVITY OF ETHANOLIC AJWAIN (TRACHYSPERMUM
AMMI) EXTRACT ON ESBL AND MBL PRODUCING UROPATHOGENS
MOBASHSHERA TARIQ, MEGHANA GORE AND ARUNA K*
Department of Microbiology, Wilson College, Mumbai 400007, India.
Email: [email protected]
Received: 21 Apr 2014 Revised and Accepted: 19 May 2014
ABSTRACT
Objective: The worldwide problem of emerging antibiotic resistance has created a need to explore alternative approaches of treatment. One such
approach is based on evaluating herbal compounds for their activity against pathogens causing infections.
Methods: In the current study, the effect of ethanolic extracts of ajwain was studied on 7 Metallo β-lactamase and 50 Extended Spectrum βlactamase producing uropathogens which were identified and characterized previously in our laboratory. The bioactive components from ajwain
were extracted at 60°C for 6-8h with the help of soxhlet apparatus using ethanol as solvent. This extract was concentrated at 40°C on water bath to
obtain a semisolid mass which was used to perform qualitative and quantitative analysis.
Results: Bioassay of ethanolic extracts from ajwain showed 14mm-21mm zones of inhibition for ESBL and MBL producers. Minimum Bactericidal
Concentrations (MBC) of ajwain extracts were found to be in the range of 0.5-10mg/ml. Ajwain extracts also showed synergistic activity with
ampicillin by lowering the MBC of ampicillin from 10mg/ml to 200-400µg/ml. Time kill analysis of ajwain extracts showed complete loss of viability
of test cultures after 2h incubation at 37⁰C at MBC concentrations and significant decrease in viability after 4h incubation at 37⁰C at ½MBC
concentrations. Gas Chromatography Mass Spectroscopic analysis of the ajwain extracts showed the presence of iso-thymol (carvacrol) as its major
constituent. Other components like cymene, terpinene, terpineol, emersol, ethyl ester etc were also detected.
Conclusion: These results collectively indicate the possible use of ajwain extracts in combination therapy to treat infectious diseases caused by
multiple drug resistant pathogens.
Keywords: ESBL, MBL, Ajwain, Antibiotic resistance, Bioassay.
INTRODUCTION
Urinary tract infections (UTIs) are the most frequent bacterial infections
encountered in community settings [1, 2]. It is estimated that a person is
infected with UTI at-least once in his/her lifetime [3]. The most
commonly encountered gram negative uropathogens are E.coli,
K.pneumoniae, Citrobacter spp, P.aeruginosa and Proteus spp [4]. Of late,
treatment of these common infectious diseases has become difficult due
to the emergence of antibiotic resistant strains like ESBL (Extended
spectrum β-lactamase) and MBL (Metallo- β-lactamase) producers.
Extended spectrum β-lactamases (ESBLs) are enzymes produced by
pathogenic bacteria that are capable of hydrolyzing oxyiminocephalosporins, and are inhibited by β-lactamase inhibitors [5].
MBLs are bacterial zinc enzymes that are able to hydrolyze most βlactam antibiotics [6, 7]. In addition, they also show a high degree of
resistance to other groups of antibiotics [8]. The emergence of everincreasing Multiple Drug Resistant (MDR) microbial strains has
become a severe health threat to human-kind, and one of the biggest
challenges to global drug discovery programs [9, 10]. In order to
control such highly infectious cases, medical practitioners are left
with no choice but to use higher doses of antibiotics, combination
therapy etc. To avoid the present scenario of extreme drug
resistance, the focus is gradually shifting towards using natural
compounds from plants for treatment of infectious diseases. Plants
are rich in a wide variety of secondary metabolites like tannins,
terpenoids and alkaloids that have been found to have antibacterial
properties in vitro [11]. Herbal medicines have always been a rich
source of drug discovery programs, and many plant derived
compounds have shown promising activity against MDR pathogens
[12, 13]. Several studies have reported antibacterial activities of
Eriobotrya japonica, Zataria multiflora and Terminalia chebula on
ESBL and MBL producers respectively [11, 14, 15]. Plants have been
evaluated not only for their inherent antimicrobial activity, but also for
their action as a resistance-modifying agent [16, 17, 18]. The
enhancement of antimicrobial activity or the reversal of antibiotic
resistance by natural or synthetic non-conventional antibiotics has led
to the classification of these compounds as enhancers of antibiotic
activity [19]. A recent study has shown synergistic activity of
Pomegranate pericarp extracts with ciprofloxacin on ESBL and MBL
producers [20]. Trachyspermum ammi, commonly known as ajowan or
ajwain, is commonly used as a spice. It is a highly valued and
medicinally important seed-spice of the family Apiaceae grown in Iran,
India, Pakistan and Egypt. It has been used as culinary spice
worldwide and resembles thyme. It was traditionally used as a food
flavouring agent and as a digestive stimulant [21].
Since ancient times, it has been used as a therapeutic agent against
inflammatory diseases. It is known to have antibacterial [22],
antifungal [23], antiseptic [24], anti-helminthic [25], hypolipidaemic
[26], antioxidant [27], anti-inflammatory [28] and carminative [29]
properties. It was mainly used by the practitioners of the ayurvedic
and unani systems of medicine for the treatment of several
disorders. It is still used as an expectorant [30], galactogogue [31],
antitussive [32] and diurectic [33] agent in many developing
countries. The active components of ajwain oil are phenols, mainly
thymol and some carvacrol [21]. Thymol is a monopterone
compound, which gives aromatic fragrance to seeds. It has local
anesthetic, anti-bacterial and antifungal properties. Thymol and
Carvacrol inhibit the peroxidation of liposome phospholipids in a
concentration dependent manner [34]. It also provides antiviral,
antihypertensive, antispasmodic, bronchodilator, and hepato
protective properties in addition to the previously described
benefits of ajwain [35].
In addition, ajwain also contains small amounts of other phytochemicals such as pinene, cymene, limonene and terpinene. The
presence of terpenes, glycosides and sterols in plant has been found
to possess anti-inflammatory properties [21]. The phenolic
constituents of ajwain are mainly responsible for the antiseptic and
antitussive properties [21]. In India, ajwain seeds are used to ease
asthma [36].
Aruna et al.
The current study was carried out to investigate the bactericidal and
synergistic effect of ajwain extracts with ampicillin on ESBL and
MBL producing uropathogens.
MATERIALS AND METHODS
Test organisms
195 gram-negative uropathogens were collected from local
pathological laboratories and hospitals situated in south Mumbai
Int J Pharm Pharm Sci, Vol 6, Issue 6, 278-284
and were characterized for ESBL and MBL production in our
laboratory in a previous study [37].
Fifty ESBL producing uropathogens that included 10 representative
isolates of each of the following genera, i.e., Klebsiella, Escherichia,
Pseudomonas, Proteus and Citrobacter, and 7 MBL producing uropathogens
were used in the current study. They are listed in Table 1. These isolates
were maintained on Luria-Bertani (LB) agar slants supplemented with
100μg/ml of ampicillin and stored at refrigerated conditions.
Table 1: Test Pathogens used in the study
Organism
E.coli
Klebsiella pneumoniae
Citrobacter amalonaticus
Citrobacter diversus
Pseudomonas aeruginosa
Proteus mirabilis
Proteus vulgaris
Total
ESBL Producers
Total no.
10
10
3
7
10
5
5
50
Processing of the Ajwain seeds
Ajwain seeds were purchased from the local market and
authenticated by an expert botanist. It was then used for the
extraction of its bioactive components. The dried seeds were
crushed into fine powder with the help of a mechanical grinder and
refrigerated in sealed vials until further use.
Preparation of the extract
Over 100g of the processed ajwain powder was extracted with
200ml of ethanol using soxhlet apparatus for a period of 6-8h. The
extract was further concentrated at 40°C on a water bath to obtain a
semisolid mass. This mass was re-suspended in ethanol to get the
required concentration of the extract for carrying out further
analysis. This concentrated extract was prepared in large volume
and preserved at 4⁰C in sealed vials until further use. This procedure
avoids batch to batch variations.
Sterility testing of Plant extracts
The sterility of ajwain extract was checked by inoculating a loopfull
of the extract on Nutrient Agar (NA) and Sabouraud’s Agar (SAB)
plates, and checking for growth of bacterial and fungal contaminants
respectively after 1 week of incubation at room temperature [38].
Qualitative evaluation of antibacterial efficacy of ajwain extract
by agar well diffusion method
The effect of ethanolic extracts of ajwain on the test pathogens was
assayed by agar well diffusion method [39]. A loopfull of each of the test
isolates were inoculated in 10 ml of Brain Heart infusion (BHI) broth and
incubated at 37°C for 24h in order to obtain actively growing log phase
cultures. Sterile 20 ml of molten NA butt was cooled to around 40°C and
then seeded with 0.4 ml test culture (0.1 O.D. at 540nm) and poured into
sterile Petri plates. Using a sterile cork borer (8mm diameter), wells was
punched in each plate after solidification of the medium. 50µl of the plant
extract was then added to the wells, and incubated at 37°C for 24h to
observe the zones of inhibition against the extract. Control wells were
also set up using 50µl of ethanol (solvent) for each isolate. The
experiment was carried out in triplicates, and the results were reported
as mean ± Standard Deviation (SD).
Evaluation of MBC of ajwain and ampicillin by agar dilution
method
The agar dilution method was used to determine the MBC of ajwain
extract and ampicillin individually. Different concentrations of
ethanolic extracts of ajwain (0.5-10 mg/ml with an interval of 0.5
mg/ml) or ampicillin (1-10mg/ml with an interval of 1mg/ml) were
supplemented into molten NA butts cooled to around 40°C. After
solidification of the medium, 5µl of test isolates were spot-
MBL Producers
Organism
E.coli strain 1
E.coli strain 2
E.coli strain 3
Klebsiella pneumoniae strain 1
Klebsiella pneumoniae strain 2
Citrobacter amalonaticus
Pseudomonas aeruginosa
Total
Total no.
1
1
1
1
1
1
1
07
inoculated on the plates, and incubated at 37°C for 24h. MBC was
defined as the lowest concentration of plant extract/ampicillin that
completely inhibited the growth of test cultures [40].
Determination of the synergistic action by agar dilution method
The agar dilution method was similarly used to determine the
synergistic activity between ajwain extracts and ampicillin. It was
done by incorporating sub-lethal (½MBC) concentrations of ajwain
extracts into molten NA butt which were cooled to around 40°C along
with 100-500 µg/ml of ampicillin with an interval of 100µg/ml [40].
Time Kill Analysis
A time kill analysis was carried out by performing a viable count of test
pathogens in the presence of MBC and 1/2 MBC concentrations of
ajwain extract in terms of CFU/ml. The surviving pathogens were
estimated over a period of 4h. The time kill analysis was performed
using method described by Carson et al [41]. The stock concentration
of ajwain extract was prepared in nutrient broth while MBC and ½
MBC concentrations (50ml each) were prepared in the respective
broth medium from the stock concentration. The final concentration of
test microorganism in each tube was around 105 CFU/ml, which was
confirmed by viable count method. The nutrient broth medium
without ajwain extract was used as a control. 0.5ml of sample aliquots
were removed at 0-4 h with an interval of 2h serially diluted and
plated on NA plates. The plates were then incubated at 37°C for 24h to
determine the effect of different concentrations of ajwain extracts on
the viability of the test organisms.
GC-MS Analysis
The bioactive components from ajwain seed extract were analyzed
by GC-MS HP 7890 system (Agilent technologies). The GC-system
was equipped with capillary column with dimensions 30m X
0.25mm X 0.25μm. The program used for GC oven temperature was
5 min isothermal at 300ºC, followed by 90º-260ºC at a rate of
10ºC/min, then held at 260ºC for 5 min, The injection port
temperature was 240º C [42]. The used was Joel, AccuTOF GCV MS
system with time of flight analyzer was used. The entire analysis was
carried out at IIT Bombay, Mumbai 400076. The compounds of the
crude extract were identified by comparison of their retention
indices (RI) and mass spectra fragmentation with those on the
stored library available with IIT, Bombay.
RESULTS
Sterility testing of Plant extracts
Ethanolic extracts of ajwain was found to be free from bacterial and
fungal contaminants as observed by streaking it on NA and SAB
plates after 1 week of incubation at room temperature.
279
Aruna et al.
Qualitative evaluation of antibacterial efficacy of ajwain extract
by agar well diffusion method
Figure 1 illustrates the antibacterial activity of ethanolic extracts of
ajwain. The mean zones of inhibition were found to be in the range of 1520 mm (Table 2) for all the tested ESBL and MBL producing isolates.
Evaluation of MBC of ajwain extracts and ampicillin individually
and synergistically by agar dilution method
The MBC of ajwain extracts and ampicillin were estimated by agar
dilution method. The MBC values are tabulated in Table 3. All the test
pathogens showed very high MBC for ampicillin (>10mg/ml). The MBC
of ajwain extracts was found in the range of 20-30mg/ml (Table 3).
Int J Pharm Pharm Sci, Vol 6, Issue 6, 278-284
However, in presence of sub-lethal concentrations of ajwain extract,
the MBC of ampicillin was significantly reduced to 200-400μg/ml.
This demonstrates the synergistic effect between ajwain extract and
ampicillin
GC-MS analysis
The GC-MS analysis identified 7 major compounds in ethanolic
ajwain extract; they are listed in Table 4. It was observed that
carvacrol (iso-thymol) was present in the highest concentration in
ajwain extract followed by emersol. Other components like Phenol
4methoxy 2, 3- 6 trimethyl, Ethyl ester, Terpineol, Terpinene and
Cymene were present in small concentrations. The retention times
of the identified components are indicated in Table 4.
1
b
2
1
2
a
Fig. 1: Well diffusion test showing zones of inhibition observed for ESBL producing E.coli (a) and MBL producing Citrobacter amalonaticus
(b). In the figure, 1 and 2 denotes control (50μl ethanol) and Test (50μl ethanolic ajwain extract) respectively.
Table 2: Well diffusion test showing zones of inhibition observed against test pathogens by ajwain extracts
Test pathogens
ESBL Producers
E.coli
Klebsiella pneumoniae
Citrobacter amalonaticus
Citrobacter diversus
Pseudomonas aeruginosa
Proteus mirabilis
Proteus vulgaris
MBL producers
E.coli strain 1
E.coli strain 2
E.coli strain 3
Klebsiella pneumoniae strain 1
Klebsiella pneumoniae strain 2
Citrobacter amalonaticus
Pseudomonas aeruginosa
No. of isolates
Mean zones of inhibition (mm) ± SD (n=3)
10
10
3
7
10
5
5
17.67 ± 0.40
15.72 ± 0.58
18.67 ± 0.57
17.33 ± 0.39
15.33 ± 0.67
18.67 ± 0.63
19.84 ± 0.52
1
1
1
1
1
1
1
15.33 ± 0.57
19.67 ± 0.57
15.33 ± 0.57
18.33 ± 0.57
18.67 ± 0.57
15.33 ± 0.57
16.33 ± 0.57
Table 3: Minimum Bactericidal Concentration of ampicillin and ajwain extracts individually and synergistically against the test pathogens
Test pathogens
ESBL Producers
E.coli
Klebsiella pneumoniae
Citrobacter amalonaticus
Citrobacter diversus
Pseudomonas aeruginosa
Proteus mirabilis
Proteus vulgaris
MBL Producers
E.coli strain 1
E.coli strain 2
E.coli strain 3
Klebsiella pneumoniae strain 1
Klebsiella pneumoniae strain 2
Citrobacter amalonaticus
Pseudomonas aeruginosa
No. of
isolates
MBC of
ampicillin
MBC of Ajwain
extracts
Synergism:- MBC of ampicillin in
presence of sub-lethal concentration of
Ajwain extract.
10
10
3
7
10
5
5
> 10
mg/ml
1.5-5mg/ml
1.5-2.5mg/ml
1-2.5mg/ml
1-2.5mg/ml
1-10mg/ml
1mg/ml
1mg/ml
200μg/ml
300-400μg/ml
300-500μg/ml
300-500μg/ml
300-400μg/ml
300-400μg/ml
300-400μg/ml
1
1
1
1
1
1
1
> 10
mg/ml
10mg/ml
10mg/ml
5mg/ml
1.5mg/ml
1.5mg/ml
1.5mg/ml
2.5mg/ml
200μg/ml
200μg/ml
200μg/ml
200μg/ml
200μg/ml
200μg/ml
200μg/ml
280
Aruna et al.
Int J Pharm Pharm Sci, Vol 6, Issue 6, 278-284
Table 4: Components identified from ethanolic ajwain extract by GC-MS analysis
S. No.
1
2
3
4
5
6
7
Retention time
3.9
4.7
5.5
10.1
22.7
22.8
27.9
Compound
Cymene
Terpinene
Terpineol
Iso-thymol (carvacrol)
Emersol
Ethyl ester
Phenol 4methoxy 2, 3- 6 trimethyl
Time kill analysis
The time kill curves of 4 ESBL and 4 MBL producers are shown in
figures 2a, 2b, 2c, 2d and 3a, 3b, 3c, 3d respectively. All isolates
showed complete loss of viability within 2 h of growth at MBC
concentration for both ESBL and MBL producers. A significant
reduction in viability was observed at ½ MBC concentrations
gradually over 4h time period analyzed.
D
Fig. 2: Time kill curves of ESBL producing (a) E.coli (b)
K.pneumoniae (c) P.aeruginosa and (d) Citrobacter diversus
over 4h at MBC and ½ MBC.
A
A
B
C
B
281
Aruna et al.
Int J Pharm Pharm Sci, Vol 6, Issue 6, 278-284
and MBL producing pathogens at concentrations ranging from 110mg/ml. Since the extract used in the current study was prepared by
simple crude extraction using a soxhlet apparatus, the potency of the
extract can be increased by introducing modifications in the extraction
system and choice of appropriate solvents.
C
D
Fig. 3: Time kill curves of MBL producing (a) K.pneumoniae
strain 1 (b) Citrobacter amalonaticus (c) P.aeruginosa and (d)
K.pneumoniae strain 2 over 4h at MBC and ½ MBC.
DISCUSSION
Qualitative as well as quantitative assays of bacterial inhibition
using several plant extracts have been carried out by various
scientists [43, 44, 45, 46, 47]. However, most of the studies deal with
the effect of the extract on general, non-pathogenic bacteria. Very
little data is available on its benefits in treating pathogenic, drugresistant bacteria [48, 49, 50]. Although the antibacterial and
antifungal activity of ajwain is reported [51, 52, 53, 54, 55], the
amount of data published about its effectiveness against drug
resistant pathogens is very scanty [56]. To that extent, the present
study was focused on the bactericidal as well as synergistic activity of
ethanolic ajwain extracts with ampicillin. Qualitative antibacterial
analysis of ajwain extracts was carried out by the well diffusion
method.
The ajwain extracts showed prominent zones of inhibition of about
15-19mm in diameter against ESBL and MBL producing uropathogens.
Several other antimicrobial studies of ajwain done on sensitive isolates
have shown similar activities [57, 58]. It is known that herbal extracts
provide similar activity against drug resistant as well as drug sensitive
organisms [59, 60, 61, 62, 63, 64, 65]. This may be due to the
difference in modes of action of various compounds present in the
extracts, to which the organism was never exposed before and hence
never had a chance to develop resistance. Another possibility is that
the presence of multiple bioactive components in the crude extracts
may exhibit synergistic activity collectively against the pathogens.
Whereas the development of resistance against one single compound
is comparatively easier, the development of resistance against
combined action of multiple compounds is difficult.
Quantitative antibacterial analysis of ethanolic ajwain extracts, evaluated
by determining MBC showed complete inhibition of the growth of ESBL
More than 99% reduction in the MBC of ampicillin was observed in
the presence of sub-lethal (1/2 MBC) concentration of ajwain
extracts, hence indicating promising synergistic activity between
ajwain extracts and ampicillin. Herbal medicines have always been a
rich source of drug discovery programs. Many plant derived
compounds have shown promising activity against MDR bacteria
and have caused a reversal of antibiotic resistance due to its
synergistic interaction with the drug (10, 12, 13, 48, 49]. Plant
antimicrobials have been found to be synergistic enhancers in that
though they may not have any antimicrobial properties individually,
but when they are taken concurrently with the standard drug, they
enhance the activity of the drug [48].
Several published data show synergistic activities between plant
extracts and antibiotics [66, 67, 68]. To our knowledge, there is no
published data that deals specifically with the synergistic activity of
ajwain and ampicillin. Ampicillin remains the most important
antibiotic to be used for common infections, the reason being its
broad spectrum activity, low production cost and comparatively
negligible side effects [69]. However, the development of resistance
against beta-lactam antibiotics including ampicillin limits its use as a
therapeutic agent. With the reversal of antibiotic resistance by
ajwain extracts, ampicillin can be safely used even against drugresistant pathogens. The use of antimicrobial agents displaying
synergy is one of the well-established indications for combination
antimicrobial therapy. Combinations of antimicrobials that
demonstrate an in vitro synergistic effect against infecting strains
are more likely to result in successful therapeutic result. In addition,
combinations of agents that exhibit synergy or partial synergy could
potentially improve the outcome for patients with difficult to treat
infections [49]. Thus, evidence of in vitro synergism could be useful
in selecting more favourable combinations of antimicrobials for the
practical therapy of serious bacterial infections.
Our results revealed that the combined use of plant extracts and
antibiotics could be useful in the treatment of infectious diseases,
and in fighting the problem of emerging drug resistance. In-vivo
experiments, however, are needed to confirm the phenomenon of
synergy between drugs and plant extracts.
A time kill analysis of ethanolic ajwain extract against ESBL and MBL
producing uropathogens showed a significant reduction in their
viability over a 4h time period at ½ MBC concentrations. A complete
loss of viability was observed within 2h of incubation of the
pathogen at MBC concentrations. The growth curves of pathogens,
grown in the presence of ajwain extracts, indicate that the activity of
ajwain is not just bacteriostatic but also bactericidal. Thymol and
carvacrol are considered to be the active antimicrobial components
of ajwain. They act as either bactericidal or bacteriostatic depending
on the concentration used [44].
Phytochemical analysis of ajwain extract by GC-MS showed the
presence of carvacrol as its major constituent. The major component
of ajwain is thymol followed by carvacrol [70, 71, 72]. However
thymol was not detected in the extract as shown in Table 4. This may
be due its fragmentation in the harsh program conditions of GC-MS.
Another reason could be the possible obscuring of the thymol peak
in comparison with carvacrol in the chromatogram. In any case, the
bactericidal activity of ajwain and synergistic activity between
ajwain and ampicillin can be attributed to carvacrol and other
phytochemicals present in minor concentrations in ajwain.
The ethanolic extracts of ajwain showed significant activity against
ESBL and MBL producing uropathogens. Further, its potency can be
evaluated against other pathogenic drug resistant bacteria causing
other infections. Moreover, the ability of crude extracts to give such
promising antibacterial results only enhance the chances of it
becoming an important alternate remedy towards the treatment of
serious infections.
282
Aruna et al.
CONCLUSION
In the present study, the effectiveness of ethanolic ajwain extracts
was investigated against ESBL and MBL producing pathogens. These
results confirm the potential of ajwain to be used alone and in
synergy with ampicillin against ESBL and MBL producing pathogens.
18.
REFERENCES
19.
1.
20.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
Naber KG, Schito G, Botto H, Palou J, Mazzei T. Surveillance
study in Europe and Brazil on clinical aspects and
Antimicrobial Resistance Epidemiology in Females with
Cystitis (ARESC): implications for empiric therapy. European
urology 2008;54(5):1164-75.
Ronald A. The etiology of urinary tract infection: traditional
and emerging pathogens. The American journal of medicine
2002;113 Suppl 1A:14S-9S.
Saint S, Kowalski CP, Kaufman SR, Hofer TP, Kauffman CA,
Olmsted RN, et al. Preventing hospital-acquired urinary tract
infection in the United States: a national study. Clinical
infectious diseases : an official publication of the Infectious
Diseases Society of America 2008;46(2):243-50.
Singhal A, Sharma R, Jain M, Vyas L. Hospital and Community
Isolates of Uropathogens and their Antibiotic Sensitivity
Pattern from a Tertiary Care Hospital in North West India.
Annals of medical and health sciences research 2014;4(1):51-6.
Bush K, Jacoby GA, Medeiros AA. A functional classification
scheme for beta-lactamases and its correlation with molecular
structure.
Antimicrobial
agents
and
chemotherapy
1995;39(6):1211-33.
Yong D, Toleman MA, Giske CG, Cho HS, Sundman K, Lee K, et al.
Characterization of a new metallo-beta-lactamase gene,
bla(NDM-1), and a novel erythromycin esterase gene carried
on a unique genetic structure in Klebsiella pneumoniae
sequence type 14 from India. Antimicrobial agents and
chemotherapy 2009;53(12):5046-54.
Laurent P, Jose M, Nashwan N, Yvette J, Debets O. Rodríguez M,
Characterization of DIM-1, an Integron Encoded Metallo-βLactamase from Pseudomonas stutzeri clinical Isolate.
Antimicrob Agents Chemother 2010;54:2420-4.
Queenan AM, Bush K. Carbapenemases: the versatile betalactamases. Clinical microbiology reviews 2007;20(3):440-58,
table of contents.
Alanis AJ. Resistance to antibiotics: are we in the postantibiotic era? Archives of medical research 2005;36(6):697705.
Shriram V, Jahagirdar S, Latha C, Kumar V, Dhakephalkar P,
Rojatkar S, et al. Antibacterial & antiplasmid activities of
Helicteres isora L. The Indian journal of medical research
2010;132:94-9.
Suchita S, Kamat SD, Kamat DV, J. Effect of aqueous extract of
Terminalia chebula on Metallo beta-lactamase. Int Pharm Sci
2010;2:172-5.
Belofsky G, Percivill D, Lewis K, Tegos GP, Ekart J. Phenolic
metabolites of Dalea versicolor that enhance antibiotic activity
against model pathogenic bacteria. Journal of natural products
2004;67(3):481-4.
Beg AZ, Ahmad I, J. Effect of Plumbago zeylanica extract and
certain curing agents on multidrug resistance bacteria of
clinical origin. World Biot 2004;16:841-4.
Elias A, Ziad D, Abdei MR, Int J. Leaf and branch extracts of
Eriobotrya japonica exert antibacterial activity against ESBL
producing Escherichia coli and Klebsiella pneumonia.
phytomed 2011;3:120-8.
Hashemi A, Shams S, Barati M, Samedani A. Antibacterial effects
of methanolic extracts of Zataria multiflora, Myrtus communis
and Peganum harmala on Pseudomonas aeruginosa producing
ESBL. Arak University of Medical Sciences Journal
2011;14:104-12.
Kumar V, Shriram V, Mulla J, J. Antibiotic resistance reversal of
multiple drug resistant bacteria using Piper longum fruit
extract. Pharm Sci 2013;3:112-6.
Shriram V, Kumar V, Mulla J, Latha C. Curing of PlasmidMediated Antibiotic Resistance in Multi-Drug Resistant
21.
22.
23.
24.
25.
26.
27.
28.
29.
30.
31.
32.
33.
34.
35.
36.
37.
38.
39.
40.
Int J Pharm Pharm Sci, Vol 6, Issue 6, 278-284
Pathogens using Alpinia galanga Rhizome Extract. Adv Bio Tech
2013;13:01-5.
Gibbons S, Oluwatuyi M, Veitch NC, Gray AI. Bacterial
resistance modifying agents from Lycopus europaeus.
Phytochemistry 2003;62(1):83-7.
Hebshi AN, Haroni AM, Skaug N. In vitro antimicrobial and
resistance-modifying activities of aqueous crude khat extracts
against oral microorganisms. Arch Oral Biol 2006;51:183-8.
Dey D, Debnath S, Hazra S, Ghosh S, Ray R, Hazra B.
Pomegranate pericarp extract enhances the antibacterial
activity of ciprofloxacin against extended-spectrum βlactamase (ESBL) and metallo-β-lactamase (MBL) producing
Gram-negative bacilli. Food and chemical toxicology : an
international journal published for the British Industrial
Biological Research Association 2012;50(12):4302-9.
Jeet K, Devi N, Thakur N, Tomar S, Shalta L, Thakur R.
Trachyspermum ammi: A comprehensive review. Int Res J
Pharm 2012;3:133-138.
Katasani D, Srinu B, Bala R. Phytochemical screening,
quantitative estimation of total Phenolic, flavanoids and
antimicrobial evaluation of Trachyspermum ammi. Journal of
Atoms and Molecules 2011;1:1-8.
Singh DB, Singh SP, Gupta RC, T. Anti-fungal effect of volatiles
from seeds of some Umbelliferae. Mycol Soc 1979;73:349-50.
Choudhury S, Riyazuddin A, Kanjilal PA, B. P, J. Composition of
the seed oil of Trachyspermum ammi (L.) Sprague from
Northeast India. Oil Res 1998;10:588-90.
Iqbal Z, Lateef M, Khan MN, Jabbar A, Akhtar MS. Anthelmintic
activity of Swertia chirata against gastrointestinal nematodes
of sheep. Fitoterapia 2006;77(6):463-5.
Javed I, Iqbal Z, Rahman ZU, Khan FH, Muhammad F, Aslam B,
et al. Comparative anti-hyperlipidaemic efficacy of
Trachyspermum ammi-extracts in albino rabbits. Pak
2006;26:23-9.
Thangam C, Dhananjayan R, J. Anti-inflammatory potential of
the seeds of Carum copticum Linn. Indian 2003;35:388-91.
Chialva F, Monguzzi F, Manitto P, J. Akgül A. Essential oil
constituents of Trachyspermum copticum L Link fruits Oil Res
1993;5:105-6.
Chialva F, Monguzzi F, Manitto P, Akgül A. Essential oil
constituents of Trachyspermum copticum (L.) Link fruits. J
Essent Oil Res 1993;5:105-106.
Scartezzini P, Speroni E. Review on some plants of Indian
traditional medicine with antioxidant activity. Journal of
ethnopharmacology 2000;71(1-2):23-43.
Kaur M, Kaur J, Ojha S, Mahmood A. Ethanol effects on lipid
peroxidation and glutathione-mediated defense in rat small
intestine: role of dietary fats. Alcohol (Fayetteville, N.Y.)
1998;15(1):65-9.
Boskabady MH, Jandaghi P, Kiani S, Hasanzadeh L. Antitussive
effect of Carum copticum in guinea pigs. Journal of
ethnopharmacology 2005;97(1):79-82.
Ahsan SK, Shah AH, Tanira MM, Ahmad MS, Tariq M, Ageel AM.
Studies on some herbal drugs used against kidney stones in
Saudi folk medicine. Fitoterapia 1990;61:435-58.
Aeschbach R, Löliger J, Scott BC, Murcia A, Butler J, Halliwell B,
et al. Antioxidant actions of thymol, carvacrol, 6-gingerol,
zingerone and hydroxytyrosol. Food and chemical toxicology :
an international journal published for the British Industrial
Biological Research Association 1994;32(1):31-6.
Bairwa R, Sodha RS, Rajawat BS. Trachyspermum ammi.
Pharmacognosy reviews 2012;6(11):56-60.
Indian Spices-Ajwain2013.
Aruna K, Tariq M. Prevalance of extended spectrum betalactamase production among uropathogens in south Mumbai
and its antibiogram pattern. EXCLI 2012;11:363-72.
Sule LO, Agbabiaka TO. Antibacterial effect of some plant
extracts on selected Enterobacteriaceae. Ethno botanical
leaflets 2008;12:1035-42.
Toda M, Okubo S, Hiyoshi R, Shimamura T. The bactericidal
activity of tea and coffee. Lett Appl Microbiol 1989;8:123-125.
Clinical and Laboratory Standards Institute (2006) Methods for
dilution antimicrobial susceptibility tests for bacteria that grow
283
Aruna et al.
41.
42.
43.
44.
45.
46.
47.
48.
49.
50.
51.
52.
53.
54.
55.
56.
57.
aerobically-seventh edition: approved standard M07-A7. CLSI,
Wayne, PA.
Carson CF, Mee BJ, Riley TV. Mechanism of action of Melaleuca
alternifolia (tea tree) oil on Staphylococcus aureus determined
by time-kill, lysis, leakage, and salt tolerance assays and electron
microscopy. Antimicrob Agents Chem 2000;46:1914-20.
Indian Pharmacopoeia 5th ed2007.
Cox SD, Mann CM, Markham JL, Bell HC, Gustafson JE,
Warmington JR, et al. The mode of antimicrobial action of the
essential oil of Melaleuca alternifolia (tea tree oil). Journal of
applied microbiology 2000;88(1):170-5.
Caccioni DLR, Guizzardi M, Biondi DM, J. Relationships between
volatile components of citrus fruit essential oil and
antimicrobial action on Penicillium digitatum and Penicillium
italicum. Int Microbiol 2000;88:170-5.
Tassou CC, Koutsoumanis K, Nychas GJ. Inhibition of
Salmonella enteridis and Staphylococcus aureus on nutrient
broth by mint essential oil. Food Res Int 2000;48:273-80.
Toda M, Okubo S, Hiyoshi R, Shimamura T. The bactericidal
activity of tea and coffee. Lett Appl Microbiol 1989;8:123-5.
Pednekar PP, Vakil B V, Sane RT, Datar AG. Antimicrobial
activity of essential oil of Blumea eriantha DC against skin
pathogens. Int J Pharm Pharm Sci 2012;4:296-299.
Marquez B, Neuville L, Moreau NJ, Genet J-P, dos Santos AF,
Caño de Andrade MC, et al. Multidrug resistance reversal agent
from Jatropha elliptica. Phytochemistry 2005;66(15):1804-11.
Khan R, Islam B, Akram M, Shakil S, Ahmad A, Ali SM, et al.
Antimicrobial activity of five herbal extracts against multi drug
resistant (MDR) strains of bacteria and fungus of clinical origin.
Molecules (Basel, Switzerland) 2009;14(2):586-97.
Shriram V, Jahagirdar S, Latha C, Kumar V, Puranik V, Rojatkar S, et
al. A potential plasmid-curing agent, 8-epidiosbulbin E acetate,
from Dioscorea bulbifera L. against multidrug-resistant bacteria.
International journal of antimicrobial agents 2008;32(5):405-10.
Dwivedi SK, Singh KP, Frag J. Fungi toxicity of some higher
plant products against Macrophmina phascolina (Tassi) Gold.
Flavour. American journal of therapeutics 1998;13:397-9.
Singh G, Maurya S, Catalan C, De Lampasona MP. Chemical
constituents, antifungal and antioxidative effects of ajwain
essential oil and its acetone extract. Journal of agricultural and
food chemistry 2004;52(11):3292-6.
Syed M, Sabir AW, Chaudhary FM, Bhatty MK, J. Antimicrobial
activity of essential oils of umbelliferae part II-Trachyspermum
ammi, Daucus carota, Anethum graveolens and Apium
graveolens. Pak Ind Res 1986;28:189-92.
Kaur GJ, Arora DS. Antibacterial and phytochemical screening of
Anethum graveolens, Foeniculum vulgare and Trachyspermum
ammi. BMC complementary and alternative medicine 2009;9:30.
Masih U, Shrimali R, Naqvi S. Antibacterial Activity of Acetone and
Ethanol Extracts of Cinnamon (Cinnamomum zeylanicum) and
Ajowan (Trachyspermum ammi) on four Food Spoilage Bacteria.
International Research Journal of Biological Sciences 2012;1:7-11.
Rosina K, Mohammad Z, Sadul HA, Abdul L, J. AsadK. Activity of
solvent extracts of Prosopis spicigera, Zingiber officinale and
Trachyspermum ammi against multidrug resistant bacterial
and fungal strains. Dev Ctries 2010;4:292-300.
Vivek KS, Deepali S, Deepti P, Archana S, J. Dermatophytes and
related keratinophilic fungi isolated from the soil in Gwalior
region of India and in vitro evaluation of antifungal activity of
58.
59.
60.
61.
62.
63.
64.
65.
66.
67.
68.
69.
70.
71.
72.
Int J Pharm Pharm Sci, Vol 6, Issue 6, 278-284
the selected plant extracts against these fungi. Plant Res
2013;7:2136-9.
Savita A, Sanjay G, J. In Vitro Antimicrobial Studies of
Trychyspermum ammi. Int Bio Sci 2012;3:64-8.
Rani P, Khullar N. Antimicrobial evaluation of some medicinal
plants for their anti-enteric potential against multi-drug
resistant Salmonella typhi. Phytotherapy research : PTR
2004;18(8):670-3.
Parekh J, Chanda S, J. In vitro screening of antibacterial activity
of aqueous and alcoholic extracts of various Indian plant
species against selected pathogens from Enterobacteriaceae.
Afr Res 2007;1:92-9.
Tambekar DH, Khante BS, Dahikar SB, Banginwar YS, J.
Antibacterial properties of contents of Triphala: A traditional
Indian herbal preparation. Continental 2007;1:8-12.
Shafiqur M, Anwar MN, J. Rahman, Antimicrobial activity of
crude extract obtained from the root of Plumbago zeylanica
Bangladesh. American journal of therapeutics 2007;24:73-5.
Mazumder R, Bhattacharya S, Mazumder A, Pattnaik AK,
Tiwary PM, Chaudhary S. Antidiarrhoeal evaluation of Aegle
Marmelos (Correa) Linn. root extract. Phytotherapy research :
PTR 2006;20(1):82-4.
Rahman MU, Gul S, Ejaz AO, J. Antimicrobial activities of Ferula
assafoetida oil against Gram positive and Gram negative
bacteria. Env Sci 2008;4:203-6.
Bhatnagar SS, Santapau H, Desa JD, Mamar AC, Ghadielly NC,
Solomon MJ, et al. Biological Activity of Indian Medicinal Plants:
Anti-bacterial, anti-tubercular, and anti-fungal action. Indian
Res 1961;49:799-813.
Yang Z-C, Wang B-C, Yang X-S, Wang Q, Ran L. The
synergistic activity of antibiotics combined with eight
traditional Chinese medicines against two different strains
of Staphylococcus aureus. Colloids and surfaces. B,
Biointerfaces 2005;41(2-3):79-81.
Betoni JEC, Mantovani RP, Barbosa LN, Di Stasi LC, Fernandes
Junior A. Synergism between plant extract and antimicrobial
drugs used on Staphylococcus aureus diseases. Memorias do
Instituto Oswaldo Cruz 2006;101(4):387-90.
Freitas E, Aires A, de Santos Rosa EA, Saavedra MJ.
Antibacterial activity and synergistic effect between
watercress extracts, 2-phenylethyl isothiocyanate and
antibiotics against 11 isolates of Escherichia coli from
clinical and animal source. Letters in applied microbiology
2013;57(4):266-73.
Timothy J, Vicky D, J. Management of uncomplicated urinary
tract infections. West 2002;176:51-5.
Seo S-M, Park H-M, Park I-K. Larvicidal activity of ajowan (
Trachyspermum ammi ) and Peru balsam ( Myroxylon pereira )
oils and blends of their constituents against mosquito, Aedes
aegypti , acute toxicity on water flea, Daphnia magna , and aqueous
residue. Journal of agricultural and food chemistry
2012;60(23):5909-14.
Zarshenas MM, Samani SM, Petramfar P, Moein M. Analysis of
the essential oil components from different Carum copticum L.
samples from Iran. Pharmacognosy research 2014;6(1):62-6.
Rafiul H, Ansari S, Abul KN, Kamran JN, Hptlc, J. Validated
Method For Quantification of Thymol Content In
Trachyspermum ammi and Poly herbal Unani Formulation Arq
Zeera. Int Pharm Sci 2012;4:478-82.
284